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WO1996011709A1 - Synthese du vaccin antityphoidique a partir d'un polysaccharide de plante ou de fruit - Google Patents

Synthese du vaccin antityphoidique a partir d'un polysaccharide de plante ou de fruit Download PDF

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Publication number
WO1996011709A1
WO1996011709A1 PCT/US1995/012585 US9512585W WO9611709A1 WO 1996011709 A1 WO1996011709 A1 WO 1996011709A1 US 9512585 W US9512585 W US 9512585W WO 9611709 A1 WO9611709 A1 WO 9611709A1
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WIPO (PCT)
Prior art keywords
acetylated
modified
polygalacturonate
carrier
saccharide
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PCT/US1995/012585
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English (en)
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WO1996011709B1 (fr
Inventor
Shousun Chen Szu
Slavomir Bystricky
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The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
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Application filed by The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services filed Critical The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority to EP95935689A priority Critical patent/EP0787015B1/fr
Priority to DE69525638T priority patent/DE69525638T2/de
Priority to AU37607/95A priority patent/AU3760795A/en
Priority to AT95935689T priority patent/ATE213647T1/de
Publication of WO1996011709A1 publication Critical patent/WO1996011709A1/fr
Publication of WO1996011709B1 publication Critical patent/WO1996011709B1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to immunoprophylaxis and vaccines. More particularly it relates to modifying a plant, fruit or synthetic polysaccharide such that it is immunogenic and may be used as a vaccine to prevent typhoid fever in infants and young children.
  • Typhoid fever caused by Salmonella typhi , remains a common and serious disease in many parts of the world.
  • the capsular polysaccharide (Vi) is both an essential virulence factor and a protective antigen of Salmonella typhi [19] .
  • Tac et et al. in J. Infect. Pis. 154: 342-345 (1986) disclose a vaccine made from the Vi capsular polysaccharide of Salmonella typhi .
  • Field trials in Nepal and in the Republic of South Africa showed that a single injection of Vi conferred about 70% protection against typhoid fever in older children and in adults [1,13].
  • the mechanism of its protective action is to elicit a critical level of serum antibodies.
  • the immunologic properties of the Vi that limits its use as a vaccine are: 1) only ⁇ 70% efficacy in individuals 5 to 45 years of age; 2) an age-dependent serum antibody response, Vi elicited a comparatively short-lived antibody responses in 2 to 5 year old children and only low levels of antibodies in a fraction of children ⁇ 2 years-old and; 3) reinjection did not elicit a booster antibody response (T-cell independent) '[15,19].
  • T-cell independent T-cell independent
  • the Vi was conjugated to proteins [22,24,25] .
  • a clinical trial in adults in the United States showed that Vi-protein conjugates elicited significantly higher levels of serum antibodies than the Vi alone [25] .
  • the Vi is a linear homopolymer of (1 ⁇ 4) -Q ⁇ -D- GalApNAc, variably O-acetylated at C 3 (Fig. l) [19, 23].
  • hiteside and Baker in . Immunol. 86:538-542 (1961) and Landy et al., A . J. Hyg. 73: 55-65 (1961) disclose that the O-acetyl groups on Vi is essential for its antigenicity.
  • Szu et al. disclose a conjugate scheme for Vi capsular polysaccharide covalent bound to a carrier protein (22, 23, 24).
  • synthesis of Vi-protein conjugates poses several problems.
  • the oligo- or polysaccharide is based on pectin which has been modified by acetylation at the C 2 and/or C 3 hydroxyls of its galacturonate subunit.
  • methods are provided to synthesize a modified plant, fruit or synthetic oligo- or polysaccharide which is structurally similar to the vi antigen.
  • Fig. 1 shows the structure of the repeating unit of the Vi, the pectin and the O-acetylated pectin.
  • C 2 (R) is N-acetylated and C 3 (R 1 ) is O-acetylated;
  • C 2 and C 3 are hydroxylated;
  • OAcPec C 2 and C 3 are O-acetylated, n ⁇ number of subunits.
  • Fig. 2 shows the methyl resonances on the O-acetylated pectin by 13 C NMR spectroscopy
  • Fig. 3 shows the HPLC gel filtration profile of the O-acetylated pectin -TT conjugate through Superose 6 in 0.01 sodium phosphate, 0.1M Na2S04, pH 7.0.
  • the refractive index is the upper line and the 280nm absorbance is the lower line.
  • Fig. 4 shows the antigenicity of the O-acetylated pectin compared with Vi by double immunodiffusion.
  • Center well B-260 Vi antiserum, 1) Vi, 100 ⁇ g/ml; 2) OAcPec K + form; 3) OAcPec Ca ++ form; 4) OAcPec C 2 H 5 N + form.
  • Fig. 5 shows the quantitative precipitin analysis of pectin ( ⁇ ) , OAcPec (•) and Vi (O) .
  • Fig. 6 shows the temperature dependent stability of O-acetyls on Vi(—) and O-acetylated pectin ( ) at
  • the Vi molecule of Salmonella typhi has a simple structure which is a linear polysaccharide having repeating sugar subunits.
  • the antigenicity and immunogenicity of Vi depends on its N- acetyl at C 2 and 0- acetyl at C 3 on each galacturonate subunit [19,23].
  • N- acetyl As shown for Vi and other polysaccharides, removal of the 0- acetyls removed most of the antigenicity and all of the immunogenicity of the Vi [23,26] .
  • the precise role of N- acetyl is not known as selective removal of the N-acetyl on Vi has not been accomplished.
  • the present invention mimics the simple structure of Vi by modification of plant, fruit or synthetic saccharides.
  • the modified plant, fruit or synthetic saccharides resemble Vi in antigenic.and immunogenic properties and as such they have the capacity to act as an effective vaccines against typhoid fever.
  • the present invention encompasses a modified plant, fruit or synthetic oligosaccharide or polysaccharide.
  • Oligosaccharide as defined herein is a carbohydrate containing from two to ten simple sugar subunits linked together.
  • a polysaccharide as defined herein is a carbohydrate containing more than ten simple sugar subunits linked together.
  • the present invention preferably encompasses a modified pectin or modified D- galacturonan, oligogalacturonate or polygalacturonate and mixtures thereof.
  • modified pectin or modified oligogalactunonate or polygalacturonate refers to native or naturally occurring pectin or synthetic D- galacturonan, oligogalacturonate and polygalacturonate that has been structurally altered.
  • Such structural alterations are any alterations that render the modified pectin or modified D-galacturonan, oligogalacturonate or polygalacturonate antigenically similar to the Vi antigen of Salmonella typhi .
  • the structural alterations substantially approximate the structure of the Vi antigen of S. typhi .
  • a modified pectin, D-galacturonan, oligo-, and polygalacturonate of this invention is further characterized by its ability to immunologically mimic an epitope (antigenic determinant) expressed by S. typhi .
  • Such a modified pectin, D-galacturonan, oligo-, and polygalacturonate is useful herein as a component in an inoculum for producing antibodies that immunoreact with S. typhi , and preferably immunoreact with the Vi of S. typhi .
  • the phrase "immunologically mimic" in its various grammatical forms refers to the ability of a modified pectin, modified D-galacturonan, oligogalacturonate and polygalacturonate of this invention to immunoreact with an antibody of the present invention that recognizes and binds to a native epitope on the Vi of S. typhi as defined herein.
  • a subject modified pectin, modified D-galacturonan, oligogalacturonate and polygalacturonate need not be structurally identical to the Vi antigen so long as it includes the required sterical structure and is able to elicit antibodies that react with the Vi antigen on S. typhi .
  • a subject modified pectin, modified D- galacturonan, oligogalacturonate and polygalacturonate includes any substituted analog, fragment or chemical derivative of a pectin so long as the modified pectin, modified D-galacturonan, oligogalacturonate and polygalacturonate is capable of reacting with antibodies that react with the Vi antigen. Therefore, a present modified pectin, modified D-galacturonan, oligogalacturonate and polygalacturonate can be subject to various changes that provide for certain advantages in its use.
  • substitution includes the use of a chemically derivatized residue in place of a non- derivatized residue provided that such modified pectin, modified D-galacturonan, oligogalacturonate and polygalacturonate display the requisite immunological activity.
  • “Chemical derivative” refers to a subject modified pectin, modified D-galacturonan, oligogalacturonate and polygalacturonate having one or more residues chemically derivatized by reaction of a functional side group. Additional residues may also be added for the purpose of providing a "linker” by which the modified pectin, modified D-galacturonan, oligogalacturonate and polygalacturonate of this invention can be conveniently affixed to a label or solid matrix or carrier. Labels, solid matrices and carriers that can be used with the polypeptides of this invention are described herein below.
  • the present invention encompasses a modified pectin, modified D-galacturonan, oligogalacturonate and polygalacturonate in which the monosaccharide subunit(s) have one O-acetylated carbon, preferably two O-acetylated carbons.
  • at least C 3 or C 2 is 0- acetylated.
  • C 3 and C 2 are 0- acetylated.
  • at least 50% of C 2 and C 3 are O-acetylated.
  • the Vi molecule has N-acetyl groups at position C 2 and 0-acetyl groups at position C 3 . If all of the C 2 positions have acetyl groups and all the C 3 positions on Vi contain acetyl groups, then by definition, the Vi molecule is theoretically 200% fully acetylated. In most preparations of Vi the percent acetylation varies.
  • the C 2 position is usually about 100% N-acetylated and the C 3 position is from about 60-90% O-acetylated depending on normal variation in preparations of Vi.
  • the modified pectin, D-galacturonan, oligogalacturonate and polygalacturonate of the present invention approximates the total percent acetylation of Vi.
  • modified pectin, D-galacturonan, oligogalacturonate and polygalacturate of the present invention are from about 50% to about 200% O-acetylated, preferably from about 80% to about 200% O-acetylated, more preferably from about 160% to about 190% O-acetylated.
  • the modified pectin and the modified D-galacturonan, oligogalacturonate and polygalacturate of the present invention has a molar ratio of 0-acetyl groups/mole galacturonan sufficient to elicit antibodies that bind to Vi.
  • the molar ratio may be at least 0.5 mole of O-acetyl/mole galacturonan (Gal A) , preferably at least 1.6 moles O-acetyl/mole Gal A, more preferably between about 1.6 and about 1.9 moles O- acetyl/mole Gal A. In one embodiment, the ratio is about 1.9 moles O-acetyl/mole Gal A.
  • the molecular weight of the Vi alone and as a Vi-carrier conjugate is related to its immunogenicity [16, 17, 22] .
  • the modified pectin and modified D-galacturonan, oligogalacturonate and polygalacturonate may vary in molecular weight in order to enhance its antigenicity or to enhance its immunogenicity when in a conjugate form.
  • the modified pectin and modified D-galacturonan, oligogalacturonate and polygalacturonate may have from about 2 to about 1,000 modified galacturonic subunits, preferably from about 50 to about 800, more preferably from about 200 to about 600 monosaccharide subunits.
  • the molecular weight of the modified pectin may range from about 100 to about 1,000,000, preferably from about 200,000 to about 600,000. In one embodiment the molecular weight of the modified pectin is approximately 400 kD.
  • substitutions or deletions are encompassed, such that the substitutions or deletions result in a modified pectin and modified D- galacturonan, oligogalacturonate and polygalacturonate that is antigenically similar to the Vi antigen of S. typhi .
  • naturally occurring pectin is modified as to replace the hydroxyl groups at the C 2 and C 3 positions of galacturonic acid with 0-acetyl groups.
  • the modified pectin is referred to herein as OAcPec.
  • the characteristics of OAcPec in comparison with Vi of S. typhi is as follows:
  • OAcPec has ⁇ 5% neutral sugars and Vi had a nondetectable amount.
  • the stability of OAcPec as measured by its O-acetyl content and molecular size, is similar to that of Vi.
  • the molecular size of Vi is more stable than the OAcPec probably due to the stabilizing effect of a hydrogen bond between the N-acetyl and the carboxyl of the adjacent residue [23] . Since vaccines will be stored at s3-8°C, the stability characteristic of OAcPec and Vi can be considered as similar.
  • OAcPec and vi are antigenically indistinguishable by immunodiffusion (Fig. 4) .
  • OAcPec unlike Vi, is not immunogenic in mice probably due to its lower molecular weight [16] .
  • modified pectin, and modified D-galacturonan, oligo- , and polygalacturonate-carrier conjugate is immunogenic to Vi in mammals.
  • immunogenic is meant that the modified pectin-carrier conjugate and modified D-galacturonan, oligo-, and polygalacturonate-carrier conjugate elicit the production of antibodies upon injection into mammals.
  • the antibodies elicited are capable of specifically reacting or binding to S. typhi , are capable of specifically reacting or binding to the Vi of S.
  • modified pectin and modified D-galacturonan, oligo-, and polygalacturonate-carrier conjugate of the present invention are capable of inducing a statistically significant rise of antibodies that bind to Vi (booster effect) upon reinjection.
  • Modified pectin, and modified D-galacturonan, oligogalacturonate and modified polygalacturonate have several advantages over the vi in preparing conjugates for vaccines to prevent typhoid fever.
  • Special P 3 facilities are required to culture pathogens such as S. typhi . This restricts the availability of Vi and presents safety concerns in preparing a Vi vaccine.
  • the present invention of l) pectin, D-galacturonan, oligo- and polygalacturonate are easy to obtain, safe and purification is simpler than extraction of the Vi from S.
  • modified oligo- and polygalacturonate can be measured during the synthesis of the conjugate and in the final container by a colorimetric reaction and; 3) there is no solubility problem and the yield of modified pectin, D-galacturonan, oligo- and polygalacturonate-carrier conjugates is higher than with Vi; 4) at the 4°C, the standard storage temperature of vaccines, the stability of modified pectin, D-galacturonan, oligo- and polygalacturonate is similar to that of the Vi.
  • the present invention provides method to prepare a synthetic Vi antigen from a plant, fruit or synthetic oligo- or polysaccharide and to conjugate it with a carrier in order to enhance and elicit a booster response against Salmonella typhi capsular polysaccharide.
  • pectin, D- galacturonan, oligogalacturonate, or polygalacturonate is O-acetylated at C2 and C3 positions with acetic anhydride.
  • O-acetylated pectin, D-galacturonan, oligogalacturonate, or polygalacturonate is thiolated with cystamine, or aminolated with adipic dihydrazide, diaminoesters, ethyldiamine and the like.
  • Both the thiolated and the aminolated O-acetylated pectin, D-galacturonan, oligogalacturonate, or polygalacturonate are stable, may be freeze dried, and stored in cold.
  • the thiolated intermediate may be reduced and covalently linked to a polymeric carrier containing a sulfhydro group, an N-pyridyldithio group.
  • the aminolated intermediate may be covalently linked to a polymeric carrier containing a carboxyl group through carbodiimide condensation.
  • O-acetylated pectin, D-galacturonan, oligogalacturonate, or polygalacturonate covalently linked to a polymeric carrier is immunogenic in mammals and can serve as a typhoid fever vaccine.
  • Pectin extracted and purified from plants or fruits such as, but not exclusive, inner portion of the rind of citrus fruits such as oranges, fruit pomaces as from apples or beets, and the like, can be used as the source of polysaccharide.
  • the pectin may be further purified, for example, by precipitation with ethanol or gel filtration and the like.
  • Pectin can be O-acetylated by treatment with acetic anhydride in formamide and pyridine. The content of O-acetyl groups can be increased by repeat the acetylation process until the desired level of acetylation is achieved.
  • Polymeric carriers are chosen on the basis of facilitating two functions: 1) to increase the immunogenicity of the polysaccharide and 2) antibodies raised against the carrier are medically beneficial. Carriers that fulfill these criteria are described in the art (7, 10, 22-25).
  • Polymeric carriers can be a natural or a synthetic material containing a primary or/and a secondary amino group, an azido group or a carboxyl group.
  • the carrier can be water soluble or insoluble. Examples of water soluble carriers included but are not limited to natural or synthetic peptides or proteins from bacteria or virus, e.
  • water insoluble carriers include but are not limited to are aminoalkyl-Sepharose, e. g., aminopropyl or aminohexyl Sepharose, and aminopropyl glass and the like. Other carriers may be used when an amino or carboxyl group is added through covalent linkage with a linker molecule.
  • O-acetvlated pectin D- ⁇ alacturonan. oligogalacturonate. or polygalacturonate con-iu ⁇ ated with a carrier:
  • the O-acetylated pectin, D- galacturonan, oligogalacturonate, or polygalacturonate can be covalently bound to a carrier with or without a linking molecule.
  • the O-acetylated pectin, D-galacturonan, oligogalacturonate, or polygalacturonate and carrier are mixed in the presence of carboxyl activation agent, such as carbodiimide in a choice of solvent appropriate for both the pectin, D- galacturonan, oligogalacturonate, or polygalacturonate and the carrier as are known in the art.
  • carboxyl activation agent such as carbodiimide
  • the O-acetylated plant, fruit or synthetic D- galacturonan, oligosaccharide or polysaccharide is preferably conjugated to a carrier using a linking molecule.
  • a linker or crosslinking agent as used in the present invention, is a small linear molecule having a molecular weight of approximately ⁇ 500 and is non- pyrogenic and non-toxic in the final product form (7, 10, 22-25) .
  • To conjugate with a linker or crosslinking agent either or both of the pectin, D-galacturonan, oligogalacturonate, or polygalacturonate and the carrier are covalently bound to a linker first.
  • the linkers or crosslinking agents are a homobifunctional or heterobifunctional molecules, e. g., adipic dihydrazide, ethylene diamine, cystamine,
  • N-succinimidyl-3- (2-pyridyldithio) propionate SPDP
  • N-succinimidyl N- (2-iodoacetyl) -b-alaninate-propionate SIAP
  • succinimidyl 4- (N-Maleimido-methyl) cyclohexane-1-carboxylate SMCC
  • 3,3' -dithiodipropionic acid and the like The linkers are bound to the carboxyl groups of the O-acetylated pectin, D-galacturonan, oligogalacturonate, or polygalacturonate or the carrier through carbodiimide condensation.
  • linkers are bound to the amino groups of the carrier through carbodiimide condensation or N-hydroxylsuccinimidyl ester.
  • the unbound materials are removed by gel filtration or ion exchange column depending on the materials to be separated.
  • the final conjugate consist of the oligo- or polysaccharide and the carrier bound through a linker.
  • the modified pectin-carrier conjugates and modified D-galacturonan, oligogalacturonate and polygalacturonate-carrier conjugates of the present invention elicit antibodies that react with or bind to the Vi antigen.
  • the anti-Vi antibody levels elicited by the modified pectin-carrier conjugates were comparable to those elicited by a Vi- Pseudomonas aeruginosa recombinant exoprotein A (rEPA) conjugate as measured by ELISA.
  • rEPA Vi- Pseudomonas aeruginosa recombinant exoprotein A
  • the modified pectin-carrier and modified D-galacturonan, oligogalacturonate and polygalacturonate-carrier conjugate may be used as an effective vaccine against S. typhi to prevent or ameliorate typhoid fever in humans.
  • the present inoculum contains an effective, immunogenic amount of modified pectin-carrier conjugate and modified D-galacturonan, oligogalacturonate and polygalacturonate-carrier conjugates of this invention.
  • the effective amount of modified pectin-carrier conjugate and modified D-galacturonan, oligogalacturonate and polygalacturonate-carrier per unit dose sufficient to induce an immune response to the Vi antigen depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen as is well known in the art.
  • Inocula typically contain modified pectin-carrier conjugate and modified D- galacturonan, oligogalacturonate and polygalacturonate- carrier conjugate concentrations of oligo- or polysaccharide of about 1 micrograms to about 100 milligrams per inoculation (dose) , preferably about 25 micrograms to about 50 milligrams per dose.
  • unit dose refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material (oligo- or polysaccharide) calculated to produce the desired immunogenic effect in association with the required diluent.
  • active material oligo- or polysaccharide
  • the specifications for the novel unit dose of an inoculum of this invention are dictated by and are directly dependent on (a) the unique characteristics of the active material and the particular immunologic effect to be achieved, and (b) the limitations inherent in the art of compounding such active material for immunologic use in animals, as disclosed in detail herein, these being features of the present invention.
  • Inocula are typically prepared as a solution in tolerable (acceptable) diluent such as water, saline or phosphate-buffered saline or other physiologically tolerable diluent such as water and the like to form an aqueous pharmaceutical composition.
  • tolerable (acceptable) diluent such as water, saline or phosphate-buffered saline or other physiologically tolerable diluent such as water and the like to form an aqueous pharmaceutical composition.
  • the route of inoculation may be intramuscular, sub-cutaneous and the like, which results in eliciting antibodies protective against S. typhi .
  • the dose is administered at least once.
  • a second or booster dose may be administered approximately 4 to 6 weeks after the initial injection. Subsequent doses may be administered as indicated.
  • antibody in its various grammatical form is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v) as well as chimeric antibody molecules.
  • An antibody combining site or antigen binding fragment is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) an antigen.
  • the term immunoreact in its various forms means specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.
  • An antibody of the present invention i.e., an anti-Vi antibody
  • an anti-Vi antibody in one embodiment is characterized as comprising antibody molecules that immunoreact with: l) S. typhi and 2) isolated Vi antigen of S. typhi .
  • the antibody in another embodiment is characterized as comprising antibody molecules that immunoreact with: l) S. typhi , 2 ) isolated Vi antigen of S. typhi and 3) a modified pectin of the present invention, and being substantially free of antibody molecules that immunoreact with native or naturally occurring pectin.
  • An antibody of the present invention is typically produced by immunizing a mammal with an inoculum containing a modified pectin-carrier conjugate and modified D-galacturonan, oligogalacturonate and polygalacturonate-carrier conjugates of this invention and thereby induce in the mammal, antibody molecules having immunospecificity for the immunizing conjugate. The antibody molecules are then collected from the mammal.
  • the antibody molecules of the present invention may be polyclonal or monoclonal antibody. Monoclonal antibodies may be produced by methods known in the art.
  • the antibody of the present invention may be contained in blood plasma, serum, hybridoma supernatants and the like.
  • the antibody of the present invention is isolated to the extent desired by well known techniques such as, for example, by using DEAE Sephadex.
  • the antibodies may be purified so as to obtain specific classes or subclasses of antibody such as IgM, IgG, IgA, IgG,, IgG 2 , IgG 3 , IgG 4 and the like.
  • Antibody of the IgG class are preferred for purposes of passive protection.
  • the antibodies of the present invention have a number of diagnostic and therapeutic uses.
  • the antibodies can be used as an in vitro diagnostic agent to test for the presence of S. typhi in biological samples in standard immunoassay protocols.
  • assays include, but are not limited to, radioimmunoassays, EIA, fluorescence assay, Western blot and the like.
  • the biological sample is contacted to antibodies of the present invention and a labelled second antibody is used to detect the presence of S. typhi , or the Vi antigen of S. typhi to which the antibodies are bound.
  • Such assays may be, for example, of direct format (where the labelled first antibody is reactive with the antigen) , an indirect format (where a labelled second antibody is reactive with the first antibody) , a competitive format (such as the addition of a labelled antigen) , or a sandwich format (where both labelled and unlabelled antibody are utilized) , as well as other formats described in the art.
  • the antibodies and antigen binding fragments of the present invention are useful in prevention and treatment of infections and diseases caused by S. typhi and other microorganisms that have structures immunologically similar to the Vi antigen.
  • the dosage of administered antibodies or antigen binding fragments will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like.
  • a dosage of antibodies or antigen-binding fragments which is in the range of from about 1 mg/kg to about 10 mg/kg body weight of the mammal, although a lower or higher dose may be administered.
  • the antibodies or antigen-binding fragments of the present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, lessen or attenuate the severity, extent or duration of the infection by S. typhi .
  • the administration of the agents of the invention may be for either "prophylactic” or "therapeutic" purpose.
  • the agents are provided in advance of any symptom.
  • the prophylactic administration of the agent serves to prevent or ameliorate any subsequent infection.
  • the agent is provided at (or shortly after) the onset of a symptom of infection.
  • the agent of the present invention may, thus, be provided either prior to the anticipated exposure to S. typhi (so as to attenuate the anticipated severity, duration or extent of an infection and disease symptoms) or after the initiation of the infection.
  • modified pectin, modified D-galacturonan, oligogalacturonate, polygalacturonate, alone or linked to a carrier, as well as antibodies and other necessary reagents and appropriate devices and accessories may be provided in kit form so as to be readily available and easily used.
  • Pectin GENE pectin, from Copenhagen, Denmark, type LM-1912CSZ was extracted from citrus.
  • Carboxyls were measured by the carbazole reaction with pectin as a standard [3,27]. 0- acetyl was measured with acetyl choline as a standard and the results expressed as moles/mole GalA [12] . The concentration of sulfhydryl was determined by Ellman reaction [8] . Protein was determined with BCA with BSA as a standard [20] and the content of nucleic acids was determined by A ⁇ , [28] . 13 C nuclear magnetic resonance spectroscopy (NMR) was performed with a General Electric GN300 spectrometer at room temperature [23] .
  • NMR nuclear magnetic resonance spectroscopy
  • Pectin was dissolved in PFW (10 mg/mL) at 60°C for 1 hour, cooled to room temperature and adjusted to pH 7.0 with 1M NaOH. The polysaccharide was precipitated twice with 75% ethanol and then freeze-dried. Pectin so treated contained less than 1% of protein and nucleic acid [28] . O-acetylation of pectin was performed as described [6] . Briefly, pectin (1 g) was suspended in formamide (20 mg/mL) at 50°C for 1 hour, 20 mL pyridine added, mixed and cooled to room temperature. Acetic anhydride (15 mL) was added dropwise with mixing at room temperature for 2 hours. The reaction mixture was poured into cold absolute ethanol.
  • the precipitate was filtered, dissolved in PFW and dialyzed at 3 to 8°C against multiple changes of deionized water and freeze-dried.
  • the degree of 0- acetylation was about 50%, compared to the maximum possible yield of 200%.
  • the OAcPec was subjected again to the same procedure.
  • the final product was passed through a 2.5 x 50 cm column of Sephadex G-50 in PFW and the void volume peak was passed through a sterile 0.45 micron membrane and freeze-dried. This preparation contained -1.6 moles of O-acetyl/mole GalA or 80% yield.
  • the molar content of the O-acetyl groups are determined by Hestrin reaction (12) .
  • the distribution of the O-acetyl groups are studied by the methyl resonances of 13 C NMR spectroscopy (Fig. 2) .
  • the antigenicity of the O-acetylated pectin was studied by reaction with the antiserum against Salmonella typhi in 2-dimensional immunodiffusion using Vi polysaccharide as a comparison. Immunodiffusion was performed in 1% agarose in PBS with B-260 antiserum. Quantitative precipitation was performed with 100 ⁇ L of B- 260 with equal volumes of antigen, containing 1 to 100 ⁇ g/mL, at 37°C for 1 hour and at 3-8°C for five days with occasional mixing. The precipitates were washed in cold PFS three times, dissolved in 0.8% SDS and their A- O recorded [23] . Serum Vi antibodies were measured by ELISA using a pooled hyperimmune mouse sera, quantitated by radioimmunoassay, as the standard [1] .
  • O-acetyl groups are studied at various temperatures for various periods of time.
  • OAcPec and Vi (1 mg/mL) in PBS, pH 7.0, were incubated at 3-8°C, 25°C, 37°C and 60°C. Aliquots were removed at 1, 2, and 12 wks and analyzed for their content of O-acetyl and molecular size by gel filtration.
  • Thiolation was measured on an aliquot of the polysaccharides treated with 0.1 M DTT at room temperature for 1 hour and passage through a 2.5x35 cm P10 column. Void volume fractions were titrated for their sulfhydryl content and the degree of derivatization expressed as percent cystamine.
  • SPDP in absolute ethanol, was added dropwise at room temperature with stirring to protein (5 mg/mL) in 0.15M HEPES, 0.001 M EDTA, pH 7.5 (HE buffer) to a final concentration of 0.04M. The reaction proceeded for 1 hour and dialyzed against the HE buffer overnight. The reaction mixture was passed through a 2.5x35 cm column of P10 in HE buffer and the void volume fractions concentrated to ⁇ 10 mg/mL. An aliquot was treated with 0.075 M DTT at room temperature for 2 hours and its A ⁇ used to calculate the molar ratio of SPDP to protein [5] .
  • the cystamine-derivatized polysaccharide 10 mg/mL PBS, pH 7.4, was treated with 0.05M DTT at room temperature, for two hours and passed through a 2.5x35 cm column of Sephadex G-50 in PBS, pH 7.0. An aliquot was taken to determine its sulfhydryl content and the remainder mixed with an equal weight of SPDP-derivatized protein and stirred at room temperature for 4 hours and at 3-8°C overnight. The reaction mixture was passed through a 2.5x95 cm column of Sephacryl S-1000 in PFS at 3-8°C.
  • OAcPec-TT fractions containing protein and polysaccharide were pooled into two batches: OAcPec-TT, for the void volume peak and OAcPec-TT 2 for the lower molecular weight fractions.
  • Vi-rEPA was passed through a 2.5x95 cm column of Sephacryl S-1000 in PFS and the void volume fractions pooled.
  • OAcPec 0- acetylation ranged from 0.1 to 1.6 moles/GalA for pectins. Unless specified, the OAcPec described in the following had 1.6 mole O-acetyl/mole GalA. I3 C N.M.R. of OAcPec showed more than two signals observed with acetyl methyl resonances indicating that mono and diacetylated species are present: non-O-acetylated residues could, however, be present ( Figure 2) . The stoichiometry of C 2 and C 3 0- acetylation are the same.
  • the O-acetyl groups are probably distributed equally between C 2 and C 3 (Fig. 2) , at least 60% of the GalA are di-O-acetylated, while 20% are mono-O-acetylated.
  • Neutral sugar content in the pectin is lower than 5%.
  • OAcPec similar to that of the pectin, had a broad distribution with the major peak -400 kD (Fig. 3) .
  • OAcPec was soluble in 0.15M NaCl and did not form a gel in the presence of Ca ++ .
  • Molar absorbances in the carbazole assay were l.32xl0 3 for OAcPec, 1.61xl0 3 for pectin and 1.63xl0 3 for GalA. The differences between pectin and GalA were ⁇ 2% and are probably due to neutral sugars in the pectin. Vi, in contrast, did not react in the carbazole assay.
  • Pectin did not react with B-260 serum in double immunodiffusion.
  • OAcPec in contrast, formed a line of identity with Vi (Fig. 4) .
  • Precipitation of OAcPec with Vi antiserum did not change with different counter ions including Na + , Ca ++ , K + or tetrabutylammonium.
  • pectin also yielded a line of identity with the Vi (not shown) .
  • No precipitation in double immunodiffusion was observed when the O-acetylation of pectin was s ⁇ .2 mole/mole GalA. Quantitative precipitation showed that both Vi and OAcPec precipitated 2.6 mg/mL Ab from B-260 antiserum (Fig. 5) .
  • thermostability of OAcPec and Vi O-acetyls was similar for OAcPec compared to Vi (Fig. 6) . Following storage at 3-8°C for 12 weeks, there was no change in the concentration of 0- acetyls for Vi and OAcPec compared to the original level of O-acetyls for Vi and OAcPec prior to storage: at 22°C, O-acetyls declined to 93% for Vi and to 88% for OAcPec and at 60°C, only 12% of the O-acetyls remained on Vi and 10% on OAcPec.
  • the stabilities of glycosidic linkages of the polysaccharides were studied by gel filtration. There was no change in M. of OAcPec at 3-8°C for three months. After storage of OAcPec at 60°C for three month, the M, decreased from 400 kD to 30 kD (not shown) . In contrast, the Vi was more stable: little depolymerization was observed after incubation at 60°C for two weeks and the M,. shifted from 2xl0 3 kD to 500 kD after 3 months.
  • the degree of thiolation was 4% for the O-acetylated pectin.
  • the HPLC profile of OAcPec-TT shows the conjugate and a small portion of OAcPec were eluted in the void volume (Fig. 3) .
  • the final yield of the conjugate was 20-30%.
  • the polysaccharide-protein ratio is -0.4 -0.8% wt/wt.
  • OAcPec O-acetylated pectin
  • TT tetanus toxoid
  • rEPA Pseudomonas aeru inoaa recombinant Exoprotein A
  • mice 16-20 g 9 general purpose mice from the NIH colony were injected subcutaneously 1, 2, or 3 times at 2 week intervals with 2.5 ⁇ g of the polysaccharide alone or as a conjugate. 10 mice from each group were exsanguinated two weeks after the first injection and one week after the second and third injections. Controls included mice injected with saline, Vi or OAcPec. Vi antibody levels were measured by ELISA with a reference calibrated by RIA.
  • BSA bovine serum albumin
  • Coating Buffer sodium carbonate-sodium bicarbonate buffer solution, pH 9.5 at 20°C, 30 ml 0.1M Na 2 C0 3 , 70 ml 0.1M NaHC0 3 .
  • Washing Buffer 0.85% NaCl, 0.1% Brij 35, 0.02% NaN 3 .
  • Dilution Buffer 1 x PBS, 0.1% Brij 35, 2% BSA, prepare fresh each time, filter with 0.45 ⁇ m Millipore filter.
  • Dilution buffer without BSA can be prepared as a "stock solution" and BSA added before use.
  • Substrate Buffer 1000 ml Tris-HCl 300 ml lMMgCl 2 , adjust pH to 9.8 with HCl.
  • Conjugate Buffer is the same as DB.
  • Dilution factor may be 5 to 10.
  • Optimum optical density should be between 1.0 and 1.5.
  • Vi antibodies As reported, Vi elicited serum antibodies in mice after one injection and reinjection did
  • Vi antibodies ( ⁇ g Ab/mL serum) in mice immunized with Vi, Vi-rEPA, Pectin, O-acetyl Pectin (OAcPec) and OAcPec-TT conjugates.
  • Example 6 WHO Testing Protocol of S. t ⁇ phi Vaccines
  • the modified pectin, modified D-galacturonan, oligogalacturonate and polygalacturonate-carrier conjugates vaccines are tested as per WHO requirements for acellular vaccines against Salmonella typhi (32) .
  • the following tests are carried out on each lot of modified saccharide-carrier conjugate vaccine as per WHO requirements as briefly outlined:
  • Volunteers between 18 and 45 years of age, who have no antibodies to hepatitis B and to HIV-1 are recruited. Following receipt of their informed consent, volunteers receive 1 injection of Vi (25 ⁇ g in 0.5 mL) (1,13) or l injection of a modified pectin-carrier conjugate (25 ⁇ g polysaccharide in 0.5 mL) of the present invention intramuscularly. Oral temperature is taken and the injection site of each volunteer is inspected 6, 24 and 48 hours after each injection. Volunteers receive a second injection at 6 weeks and are bled 2 weeks later and 26 weeks after the first injection. Antibodies reactive to Vi are determined by ELISA as described herein.

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Abstract

La présente invention concerne un oligosaccharide ou un polysaccharide modifié extrait de plante ou de fruit ou obtenu par synthèse et qui a été structurellement modifié pour rende le saccharide modifié antigéniquement semblable au polysaccharide capsulaire (Vi) de Salmonella Typhi. Le saccharide modifié peut se conjuguer avec un vecteur pour former un conjugué immunogène contre Salmonella Typhi. Les anticorps produits en réponse au conjugué immunogène apportent une protection contre la fièvre typhoïde. L'invention concerne également des procédés de fabrication du saccharide modifié et du conjugué immunogène.
PCT/US1995/012585 1994-10-17 1995-09-28 Synthese du vaccin antityphoidique a partir d'un polysaccharide de plante ou de fruit WO1996011709A1 (fr)

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EP95935689A EP0787015B1 (fr) 1994-10-17 1995-09-28 Synthese du vaccin antityphoidique a partir d'un polysaccharide de plante ou de fruit
DE69525638T DE69525638T2 (de) 1994-10-17 1995-09-28 Synthese eines typhusimpfstoffes unter verwendung eines polysaccharids aus einer pflanze oder einer frucht
AU37607/95A AU3760795A (en) 1994-10-17 1995-09-28 Synthesis of typhoid fever vaccine from a plant or fruit polysaccharide
AT95935689T ATE213647T1 (de) 1994-10-17 1995-09-28 Synthese eines typhusimpfstoffes unter verwendung eines polysaccharids aus einer pflanze oder einer frucht

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EP0848011A1 (fr) * 1996-12-16 1998-06-17 De Staat Der Nederlanden Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Procédé pour le couplage d'un polysaccharide et d'une protéine
US7754227B2 (en) 1998-12-04 2010-07-13 The United States Of America As Represented By The Department Of Health And Human Services Method of immunizing humans against Salmonella typhi using a Vi-rEPA conjugate vaccine
CN1645104B (zh) * 2004-12-21 2011-03-16 兰州生物制品研究所 伤寒Vi-rEPA结合疫苗中高分子结合物含量的测定方法
WO2015029056A1 (fr) 2013-08-24 2015-03-05 Bharat Biotech International Limited Vaccin bactérien et procédés de fabrication
US9475846B2 (en) 2008-06-13 2016-10-25 Glaxosmithkline Biologicals Sa Conjugated Vi saccharides

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US6258774B1 (en) * 1998-03-19 2001-07-10 University Of Medicine And Dentistry Of New Jersey Carrier for in vivo delivery of a therapeutic agent
WO2000037610A2 (fr) 1998-12-23 2000-06-29 Boyce Thompson Institute For Plant Research At Cornell Expression des antigenes de surface de l'hepatite b immunogenes dans les plantes transgeniques
CA2434668A1 (fr) 2003-07-04 2005-01-04 Laurence Mulard Nouvelle approche pour concevoir des glycopeptides a base de o-specifique polysaccharide de shigella flexneri serotype 2a
US8426567B2 (en) * 2005-08-26 2013-04-23 Econugenics, Inc. Method for enhancing mammalian immunological function
EP2386563A1 (fr) 2010-05-12 2011-11-16 Institut Pasteur Nouveaux décasaccharides O-acétylés

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0848011A1 (fr) * 1996-12-16 1998-06-17 De Staat Der Nederlanden Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Procédé pour le couplage d'un polysaccharide et d'une protéine
WO1998027107A1 (fr) * 1996-12-16 1998-06-25 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Procede de couplage de polysaccharides a des proteines
US6361777B1 (en) 1996-12-16 2002-03-26 De Staat Der Nederlanden,Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Method of coupling polysaccharides to proteins
US7754227B2 (en) 1998-12-04 2010-07-13 The United States Of America As Represented By The Department Of Health And Human Services Method of immunizing humans against Salmonella typhi using a Vi-rEPA conjugate vaccine
US8202520B2 (en) 1998-12-04 2012-06-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method of immunizing humans against Salmonella typhi using a Vi-rEPA conjugate vaccine
CN1645104B (zh) * 2004-12-21 2011-03-16 兰州生物制品研究所 伤寒Vi-rEPA结合疫苗中高分子结合物含量的测定方法
US9475846B2 (en) 2008-06-13 2016-10-25 Glaxosmithkline Biologicals Sa Conjugated Vi saccharides
WO2015029056A1 (fr) 2013-08-24 2015-03-05 Bharat Biotech International Limited Vaccin bactérien et procédés de fabrication
EP3329935A1 (fr) 2013-08-24 2018-06-06 Bharat Biotech International Limited Vaccin bactérien et procédés de fabrication
EP3335727A1 (fr) 2013-08-24 2018-06-20 Bharat Biotech International Limited Vaccin bactérien et procédés de fabrication

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KR100433986B1 (ko) 2004-09-13
KR970706845A (ko) 1997-12-01
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US5738855A (en) 1998-04-14
ATE213647T1 (de) 2002-03-15

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